Adipogenic differentiation of human mesenchymal stem cells

ABSTRACT

A composition which comprises human mesenchymal stem cells which have the potential to differentiate into cells of more than one connective tissue type and a composition which induces cells from the mesenchymal stem cell population to differentiate into the adipogenic lineage, and a process for inducing such differentiation. The composition for inducing such differentiation comprises a glucocorticoid, a compound which stimulates cAMP production or inhibits cAMP degradation (such as a phosphodiesterase inhibitor), and/or a compound which upregulates peroxisome proliferator activated receptor γ (PPAR γ) expression and/or increases its binding affinity to its DNA binding site. The process can further include isolating the adipocytes from remaining hMSCs.

This is a Divisional of application Ser. No. 09/246,003 filed Oct. 26,1998, now U.S. Pat. No. 6,322,784 which is a continuation-in-part ofapplication Ser. No. 08/700,753, filed Jul. 30, 1996, now U.S. Pat. No.5,827,740.

This invention relates to adipocytes and more particularly to producingadipocytes from human mesenchymal stem cells.

Adipose tissue provides an energy storage reserve for the body in theform of triglycerides and this tissue can release free fatty acids whencaloric intake falls below metabolic needs. In response to increaseddietary intake, the body will normally automatically increase energyexpenditure through activity to maintain an energy balance. Energy canalso be released as heat. Adipose tissue is intimately involved in themaintenance of body temperature through brown adipose tissue and energystorage through white adipose tissue. There are normal energy regulationpathways that balance dietary intake with metabolic activity largelymediated through the hypothalamus. It is now also apparent that theadipocyte plays an active role in this process and likely producesmolecules that serve to feed back and effect regulation of triglyceridemetabolism.

The two types of adipose tissue, brown and white, carry out verydifferent roles in the body. White adipose is, designed to store excesscaloric intake while brown adipose tissue uses a unique system to syphonoff excess calories and use it to generate body heat. The heat isgenerated in the mitochondria of brown adipose where oxidation ofsubstrate is utilized to create a hydrogen ion gradient that is thencollapsed in a regulated fashion generating heat instead of ATP. It hasbeen shown that transgenic animals that lack brown adipose maintainefficient metabolism, are obese and continue to overeat (Lowell et al,1993). Other rodent studies have also shown a link between obesity,continued overeating and a sensitivity to cold, suggesting a connectionto the sympathetic nervous system (Friedman and Leibel, 1992)

Imbalance in energy metabolism in the body leads to several diseasedstates, most notably obesity and obesity-induced diabetes and these canbe described as dysfunctions of energy storage tissues. A mutation inmice that leads to obesity was identified in 1950 (Ingalls et al., 1950)and the gene was recently identified by positional cloning. The productof the ob gene is a 16,000 MW protein named leptin or OB protein. Leptinis produced only by adipocytes and is a hormone which regulates thehypothalamus. A mutation has been identified in the mouse gene thatresults in premature termination of MRNA translation such that nofunctional leptin protein is made (Zhang et al. 1994). The role ofleptin in regulation of lipid metabolism is an area of intense research.Recent published investigations include studies of the upstream promoterelements found adjacent to the ob gene which have been shown to bindC/EBP (or CCAAT/enhancer binding protein) (Yeh et al, 1995 and Hwang etal., 1996). Having a model experimental system for in vitro adipogenesisof human cells would provide for discoveries in this area.

Recently it has been reported that leptin may serve as a hormone thatregulates fertility and may be the link between appropriate body weightand reproductive physiology (Chehab et al. 1996). Both underweight andoverweight women have difficulty in conceiving and this is likelyassociated with hormonal imbalance in the body of these individuals. Theconnection between body weight, fertility and the leptin produced byadipocytes has been suspected and now tested in mice. When obese mice,which normally do not produce offspring without transplanting theovaries to surrogate females, were injected with leptin, their bodyweight fell dramatically and they gave birth to their own litters(Chehab et al, 1996).

A variety of cell types have been shown to produce lipid containingvesicles under specific culture conditions. For example, mouse 3T3-L1cells derived from NIH 3T3, an immortalized mouse cell line, can begrown and cultured as a fibroblastic cell. However, after exposure todexamethasone and methyl-isobutylxanthine, the cells undergodifferentiation which results in the production of intracellularlipid-containing vacuoles (Spiegelman and Green, 1981). Rat marrowstromal cells have been shown to undergo both osteogenic and adipogenicdifferentiation when cultured with fetal calf serum and dexamethasone,but the predominating cell type varies depending on conditions(Beresford et al., 1992). Specifically, when the steroid analogdexamethasone was present throughout the time course of culture,osteogenesis was favored; but when dexamethasone was present only duringsecondary culture, the adipogenetic pathway predominated as evidenced bylineage specific markers and cytological observation. Mouse derived CH310T1/2 cells are a multipotential cell line that, when treated with5-azacytidine, undergoes terminal differentiation into adipocytes,myocytes and chondrocytes. The 5-azacytidine causes inhibition of DNAmethylation and thus causes the activation of a few genes responsiblefor commitment to these lineages (Konieczny and Emerson, 1984).

In accordance with one aspect of the present invention, there isprovided a composition and method for inducing human mesenchymal stemcells to preferentially differentiate into the adipogenic lineage, i.e.,to differentiate into adipocytes.

Applicant has found that mesenchymal stem cells (MSCs) and in particularhuman mesenchymal stem cells (hMSCs) can be directed to differentiateinto adipocytes by treating the human mesenchymal stem cells with (i) aglucocorticoid and (ii) a compound which elevates intracellular cAMPlevels by either upregulating cAMP production or by inhibitingdegradation of cAMP; in particular a compound which inhibits compound(s)which degrade cAMP.

Accordingly, in one aspect, the human mesenchymal stem cells are treatedwith a glucocorticoid and a compound which inhibits the activity of acompound which degrades cAMP; in particular a phosphodiesteraseinhibitor. The cells are subsequently cultured in media containinginsulin and fetal bovine serum.

In a preferred aspect the human mesenchymal stem cells are treated witha glucocorticoid; insulin; and at least two compounds which inhibitdegradation of cAMP, wherein one said compound which inhibitsdegradation of cAMP is indomethacin.

In a particularly preferred aspect, the human mesenchymal stem cells aretreated with a glucocorticoid; a compound which inhibits the activity ofa compound which degrades cAMP; insulin; and a compound whichupregulates peroxisome proliferator activated receptor γ(PPAR γ)expression and/or increases its binding affinity to its DNA bindingsite.

In a further embodiment, the invention provides a composition comprisingMSCs grown on a stabilized collagen gel matrix which are induced todifferentiate into adipocytes.

Human mesenchymal stem cells, as well as their isolation and expansion,have been described in U.S. Pat. No. 5,486,359. As known in the art,human mesenchymal stem cells are capable of producing two or moredifferent types (lineages) of mesenchymal cells or tissues and inparticular connective tissue. The present invention provides a methodfor generating adipocytes from primary human mesenchymal stem cells in apredictable and reproducible manner. The invention is unique in that itinvolves human cells in primary and passaged cultures rather thantransformed or immortalized cell lines that are predetermined to enterthe adipogenic pathway. hMSCs are capable of entering multiple lineagesincluding the osteocytic, chondrocytic, myocytic, tendonocytic andstromogenic lineages and the present invention provides a method andcomposition for inducing hMSCs to differentiate into adipocytes. In apreferred aspect, in accordance with the present invention, hMSC's areinduced to differentiate into essentially only adipocytes, i.e., thereis no essential production or commitment to cells of other mesenchymallineages. The method may also be used for generating adipocytes fromMSCs from other species such as rabbit, dog, rat and mouse.

The invention also provides methods to purify the adipocytes to obtain ahighly purified population.

The method of the invention for the in vitro differentiation of humanmesenchymal stem cells preferably derived from bone marrow intoadipoblastic or adipocytic cells is useful to investigators wishing tostudy this developmental program in human cells in vitro. A betterunderstanding of diseases of energy metabolism including obesity andobesity-related diabetes will also result from studies of thedifferentiation of mesenchymal stem cells to adipocytes. While acellular and biochemical basis for obesity has long been suspected,advancements have been slow due to a lack of model systems withbiochemical and molecular tools for study. Recent dramatic breakthroughsin the molecular basis of adipogenesis have opened new avenues towardsunderstanding this pathway of mesenchymal cell differentiation, althougha human model system such as the one described here has been lacking.The method will also have utility in the isolation and preparation ofadipocytes for implantation into a patient for the purpose of tissueaugmentation following trauma or cosmetic surgery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show that when human MSCs are treated in accordance with theinvention, they undergo differentiation to the adipogenic lineage. FIG.1A shows hMSCs (4×) cultured in normal hMSC media for the same period oftime as FIG. 1B There is no evidence of lipid containing vacuoles andthe cells maintain the appearance of fibroblasts at high density. InFIG. 1B are hMSCs that were allowed to become confluent and thenmaintained in normal media for 10 days prior to adding the AdipogenicInduction media (containing methylisobutylxanthine and dexamethasone)for 48 hrs, and then changed to the insulin-containing adipocytemaintenance media for an additional 2 weeks. The lipid vacuoles arefirst apparent at about 5-7 days but increase in size and abundance overtime.

FIG. 2A shows a similar control culture as FIG. 1A at highermagnification (20×). FIG. 2B show a culture of confluent hMSCs that weresubjected to Adipogenic Induction media for 48 hours and then maintainedin the Adipogenic Maintenance media for 14 days. The many lipidcontaining vacuoles of adipocytes are evident in a large proportion ofthe cells.

FIG. 3 shows the results of culturing hMSCs under a variety ofconditions, only one of which shows a high degree of adipogenicdifferentiation. All photos are at 1O× magnification. FIG. 3A shows aculture of hMSCs maintained in normal hMSC culture media alone. Thecells grow with a fibroblastic morphology. FIG. 3B shows a similarculture that was treated with Adipogenic Induction media for 48 hoursand then with Adipogenic Maintenance media for an additional 14 dayswith media changes every 3 days. The adipogenic cells, perhaps as manyas 30-35% of the cells, are evident as they contain the large refractilelipid vacuoles. FIG. 3C shows a culture of hMSCs that were maintained inthe Adipogenic Maintenance media for 14 days but was never subjected tothe dexamethasone/methyl isobutylxanthine treatment. The cells maintaina flat morphological appearance with no evident vacuoles. FIG. 3D showsa culture of hMSCs that were treated with normal hMSC media containing 1μM dexamethasone for 48 hours and then cultured for 14 days in theAdipogenic Maintenance media. The cells are disorganized but show veryfew, if any, lipid vaculoes. FIG. 3E shows a culture of hMSCs that wastreated with normal hMSC media containing 0.5 m. methyl-isobutylxanthinefor 48 hours and then was maintained for 14 days in the AdipogenicMaintenance media. The cells retain a flat fibroblastic phenotype. FIG.3F shows a culture of hMSCs that was treated with a media that inducesthe cells to differentiate along a osteogenic pathway. This mediacontains 0.1 μM dexamethasone, 10 mM β-glycerol phosphate and 50 μMascorbic acid 2-phosphate. The presence of refractile osteoid materialis evident but no large lipid vacuoles.

FIG. 4A shows a culture of hMSCs subjected to the Adipogenic Inductionmedia for 48 hours and then cultured for 14 days in the AdipogenicMaintenance media. The large lipid vacuoles are evident in this brightfield image. The lipids can also be revealed by using a fluorescentlipid soluble dye, such as Nile Red, and viewing by epifluorescenceillumination as shown in FIG. 4B. Thus, the adipogenic cells can also beidentified using vital dyes and histological stains that label the lipidvacuoles.

FIG. 5A shows hMSCs in culture which were not treated with AdipogenicInduction media but which were cultured, fixed and stained at the sametime as the adipogenic cultures shown in 5B and SC.

FIG. 5B shows hMSCs that were treated once for 48 hours with AdipogenicInduction media and then cultured for an additional three weeks inAdipogenic Maintenance media and then fixed in neutral buffered formalinand stained with Oil Red O, a lipid soluble dye that accumulates in thefat droplets.

FIG. 5C shows a dish of hMSCs that was retreated with fresh AdipogenicInduction media for a second and third 48 hour period to induce morehMSCs to become adipogenic. As many as 30-40% of the cells wereconverted to adipocytes by the three induction treatments when viewedtwo weeks after the third treatment. Panels 5A-5C were all stained withOil Red O.

FIG. 6A shows the isolated adipogenic hMSCs that attached to theuppermost surface. The population is composed of greater than 99%adipogenic cells as evidenced by the lipid droplets in every cell.

FIG. 6B shows the non-adipogenic hMSCs that settled to the lower surfaceof the flask. Very few cells containing lipid droplets were present onthe lower surface. These non-adipogenic cells could be treated withtrypsin/EDTA and replated to another dish and be shown to retainadipogenic potential (data not shown), indicating that they remain asmesenchymal stem cells, capable of lineage progression.

FIG. 7 illustrates the level of fluorescence staining of adipocytesproduced from mesenchymal stem cells cultured with varyingconcentrations of indomethacin. The numbers on the x-axis correspond tosample numbers in Table 1. The results show a dose dependent increase inthe number of adipocytes following treatment with increasing levels ofindomethacin.

FIGS. 8A-F are photographs of adipocytes produced from mesenchymal stemcells cultured with varying concentrations of indomethacin. Thephotographs show a dose dependent increase in the number of adipocytesfollowing treatment with increasing levels of indomethacin.1-methyl-3-isobutylxanthine (M) Dexamethasone (D) Insulin (I) A.Control; B. Adipocyte maintenance medium; C. MDI; D. MDI+50 μMindomethacin; E. MDI+100 μM indomethacin; F. MDI+200 μM indomethacin

FIGS. 9A-F are photographs of adipocytes produced from mesenchymal stemcells cultured with varying concentrations of indomethacin in theabsence of 1-methyl-3-isobutylxanthine. The photographs show a dosedependent increase in the number of adipocytes following treatment withincreasing levels of indomethacin, however the percentage of adipocytesproduced was much lower than using indomethacin and MDI.

A. DI+50 μM indomethacin; B. DI+100 μM indomethacin; C. DI+200 μMindomethacin; D. I+200 μM indomethacin; E. Control; F. Adipocytemaintenance medium.

FIG. 10 illustrates the level of fluorescence staining of adipocytesproduced from mesenchymal stem cells cultured with varyingconcentrations of 15-deoxy Δ^(12,14) -prostaglandin J₂ (15d-PGJ₂). Thenumbers on the x-axis correspond to sample numbers in Table 2. Theresults show a dose dependent increase in the number of adipocytesfollowing treatment with increasing levels of 15d-PGJ₂.

FIGS. 11A-F are photographs of adipocytes produced from mesenchymal stemcells cultured with varying concentrations of 15d-PGJ₂. The photographsshow a dose dependent increase in the number of adipocytes followingtreatment with increasing levels of 15d-PGJ₂. A. MDI+1 μM 15d-PGJ₂; B.MDI+10 μM 15d-PGJ₂; C. DI+1 μM 15d-PGJ₂; D. DI+10 μM 15d-PGJ₂; E. I+1 μM15d-PGJ₂; F. I+10 μM 15d-PGJ₂.

FIG. 12 is a photograph of fluorescently stained adipogenic MSCs inGELFOAM.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As noted above, one aspect of the invention provides a composition whichcomprises an isolated, homogeneous population of human mesenchymal sterncells which have the potential to differentiate into cells of more thanone mesenchymal tissue type, and a substance which induces cells fromthe mesenchymal stem cell population to differentiate into theadipogenic lineage.

In one embodiment of this aspect of the invention mesenchymal sterncells are induced to differentiate into the adipogenic lineage by use ofa glucocorticoid and at least one compound which either elevatesintracellular levels of cAMP, for example, a cAMP analog or a compoundwhich stimulates production of cAMP or inhibits degradation of cAMP; inparticular a phosphodiesterase inhibitor.

Preferred examples of the glucocorticoid are selected from the groupconsisting of dexamethasone, hydrocortisone, cortisone, etc.

Preferred examples of the substance which elevate intracellular cAMPlevels or are cAMP analogs include dibutyryl-cAMP, 8-CPT-cAMP(8-(4)-chlorophenylthio)-adenosine 3′, 5′ cyclic monophosphate;8-bromo-cAMP; dioctanoyl-cAMP, Forskolin, etc.

Preferred examples of the substance which inhibits cAMP degradation byinhibiting the activity of phosphodiesterase is selected from the groupconsisting of methyl isobutylxanthine, theophylline, caffeine andindomethacin.

The compound which elevates levels of cAMP and the glucocorticoid areused in amounts which are effective to induce hMSCs to differentiateinto adipocytes. The cAMP regulating compound and glucocorticoid may beadded to the hMSCs separately or in admixture with each other.

In general, the glucocorticoid is used in a concentration from about 0.1to 10 micromolar, preferably from about 0.5 to 2 micromolar.

When employing a compound which inhibits degradation of cAMP, such acompound is generally employed either alone or in combination withanother such compound in a concentration of about 10 to 500 micromolararid preferably from about 50 to 200 micromolar.

When employing a compound which upregulates cAMP production, suchcompound is generally employed in a concentration of from about 0.1 to100 micromolar, preferably from about 0.5 to 10 micromolar.

It is to be understood that the above amounts are representative and thescope of the invention is not to be limited thereby.

Although one of the compounds which is employed to induce hMSCs todifferentiate into adipocytes is one which regulates cAMP (either onewhich is known to upregulate cAMP production or one which preventsdegradation of cAMP), the scope of the invention is not limited to anyparticular mechanism of action. Thus, for example, even though one ofthe compounds which may be used in the present invention is aphosphodiesterase inhibitor which is known to inhibit degradation ofcAMP by inhibiting phosphodiesterase degradation of cAMP, the inventionis not limited to a mechanism of action which is dependent uponpreventing degradation of cAMP. Thus, for example, the phosphodiesteraseinhibitor may be effective for inducing differentiation of hMSCs toadipocytes by a mechanism of action other than inhibiting degradation ofcAMP.

Compounds in addition to (i) a glucocorticoid and (ii) a cAMP regulatormay be used for inducing hMSCs to differentiate into adipocytes. Thus,for example, in one embodiment insulin is also employed in conjunctionwith the cAMP regulator and glucocorticoid. In a further embodiment, acompound which upregulates the expression of peroxisome proliferatoractivated receptor γ(PPARγ) or increases the binding affinity of PPARγto its DNA binding element may also be employed in conjunction with thecAMP regulator and glucocorticoid for inducing hMSCs to differentiateinto adipocytes.

In one embodiment, there is provided a composition for inducing hMSCs todifferentiate into adipocytes which is comprised of (i) aglucocorticoid, (ii) a compound which regulates cAMP and in particular acompound which inhibits cAMP degradation such as, a phosphodiesteraseinhibitor, (iii) insulin or insulin-like growth factor and (iv) glucose.

In a preferred embodiment mesenchymal stem cells are induced todifferentiate into the adipogenic lineage by employing a glucocorticoid;insulin; and at least two compounds which inhibit degradation of cAMP,wherein one said compound which inhibits degradation of cAMP isindomethacin. In a particularly preferred embodiment, the indomethacinis used in conjunction with methyl isobutylxanthine.

In a particularly preferred embodiment of the invention, mesenchymalstem cells are induced to differentiate into the adipocytic lineage byuse of a glucocorticoid; a cAMP regulating compound; insulin; and acompound which upregulates the expression of peroxisome proliferatoractivated receptor γ(PPARγ) or increases the binding affinity of PPARγto its DNA binding element.

Representative examples of compounds that upregulate the expression ofperoxisome proliferator activated receptor γ(PPARγ) or increase thebinding affinity of PPARγ to its DNA binding element includeprostaglandins, such as members of the prostaglandin J₂ or prostaglandinD₂ families or their metabolites.

A preferred example of a substance that upregulates the expression ofperoxisome proliferator activated receptor γ(PPARγ) or increases thebinding affinity of PPARγ to its DNA binding element is 15-deoxyΔ^(12,14) -prostaglandin J₂ (15d-PGJ₂).

In general, when employing a compound that upregulates the expression ofperoxisome proliferator activated receptor γ(PPARγ) or increases thebinding affinity of PPARγ to its DNA binding element, such compound isused in a concentration of from about 0.5 to 50 micromolar andpreferably from about 1.0 to 10 micromolar.

Although in this embodiment one of the compounds employed affectsexpression of PPARγ or DNA binding of PPARγ, the scope of the inventionis not limited to the employment of this particular mechanism of action.

The addition of compounds as hereinabove described to inducedifferentiation of hMSCs to adipocytes in accordance with the inventiondoes not require that all of the treated hMSCs be induced todifferentiate into adipocytes. Thus, in accordance with an aspect of thepresent invention there is produced a composition comprised of humanmesenchymal stem cells and adipocytes wherein based on the twocomponents the adipocytes are present in an amount of at least 5 wt. %and preferably at least 15 wt. %. The amount of adipocytes may be up to50 wt. % or higher, based on the two components.

In accordance with a preferred embodiment, the composition which isgenerated is essentially free of committed cells of the mesenchymallineage other than adipocytes. In a particular preferred embodiment,there are less than one percent of committed cells of the mesenchymallineage other than adipocytes, and more preferably, less than 0.1% andmost preferably no committed cells of the mesenchymal lineage other thanadipocytes.

Although treatment of hMSCs in accordance with the invention produces amixture of adipocytes and undifferentiated hMSCs, the producedadipocytes may be recovered from the mixture to produce an isolatedpopulation of adipocytes. Representative procedures for recoveringadipocytes are described in the examples which form a part of thisapplication.

In accordance with an aspect of the present invention, hMSCs may betreated to induce differentiation into adipocytes in a manner such thatsuch differentiation is effected in vitro or in vivo.

Thus, for example, hMSCs may be admixed with compounds as hereinabovedescribed which induce differentiation into hMSCs and the resultingmixture employed in vivo to induce differentiation to adipocytes invivo. Thus, for example, the mixture without culturing in vitro for aperiod of time to induce differentiation in vitro may be employed in asuitable matrix (for example of the type hereinafter described) toinduce differentiation of the hMSCs to adipocytes in vivo.

Thus, in accordance with an aspect of the present invention there isprovided a composition comprised of human mesenchymal stem cells, aglucocorticoid and a cAMP regulator (a compound(s) which upregulatescAMP production or inhibits cAMP degradation) and a compound thatupregulates the expression of peroxisome proliferator activated receptorγ(PPARγ) or increases the binding affinity of PPARγ to its DNA bindingelement. Such a composition may be employed to produce adipocytes invitro or may be employed to induce differentiation of hMSCs in vivo.

The ability to generate large percentages of adipogenic cells from apopulation of hMSCs will allow greater numbers of cells for implantationor research studies. Fewer hMSCs would be needed as starting material.By repeating the adipogenic induction step more times, it should bepossible to induce most of the hMSCs in a population to adipocytes. Inthe case where there is a mixture of cells, adipogenic hMSCs can easilybe isolated by their buoyant density. The isolation of a highly enrichedpopulation of adipocytes from cultured hMSCs will also allow for adetailed characterization of the adipocyte phenotype

The adipocytes can be used with a variety of materials to form acomposition for purposes such as reconstructive surgery. The cells maybe combined with a biomatrix to form a two dimensional or threedimensional material as needed. Surgeons routinely use fat pads andfatty tissues from remote sites to build up an area where tissue hasbeen removed. This often involves a separate procedure with its inherentrisks.

hMSCs can also differentiate into adipocytes when cultured on threedimensional support materials to form a composite. MSCs can grow anddifferentiate on a variety of biomaterials including those made fromcollagen, polyglycolic acid, polylactic acid or copolymers thereof. Thecomposite would then be treated to induce adipogenic differentiation ofthe MSCs in vitro for 1-3 weeks, then implanted when needed. Forexample, adipogenic MSCs could be mixed with a solubilized collagen orother biomaterial which is then allowed to gel to form a threedimensional composite that could be used for breast augmentationfollowing mastectomy. Such a composite could be formed or sculpted tothe appropriate size and shape. Another composition includes theculturing of hMSCs on the acellular skin matrix that is currently on themarket such as the product by LifeCell Corporation. In this format thecells would be cultured to populate the matrix and then caused todifferentiate as described. The matrix with the adipogenic cells couldthen be cut by the surgeon to fit the site of reconstruction. As analternative hMSCs could be induced to become adipocytes prior to theirintroduction into the biocompatible materials. As another alternative,hMSCs in combination with compounds which promote differentiation intoadipocytes may be used with a biomatrix as described without culturingfor a period of time to induce differentiation whereby differentiationis induced in whole or in part in vivo.

In one embodiment, the MSCs are contacted with GELFOAM (Upjohn,Kalamazoo Mich.) as the biocompatible matrix. GELFOAM is a collagenbased material which has a sponge consistency when wet and is approvedas a hemostatic agent. Preferably the cell concentration of MSCs in theGELFOAM is in a range of from about 0.3×10⁶ to 5×10⁶ MSCs per cc ofimplant volume.

Similar to their use in reconstructive surgery, adipogenic hMSCs will beof use in elective cosmetic surgery in much the same way; to build upunderlying tissue below the skin with a composite of autologous cellsand biocompatible material.

The invention will be further described with respect to the followingexamples; however, the scope of the invention is not to be limitedthereby. Unless otherwise described percentages and parts are by weight.

Biochemical Markers of Adipocytes

A number of molecules that are specific markers of adipocytes have beendescribed in the literature that will be useful to characterize theadipocytes derived from hMSCs. These include enzymes involved in theinterconversion of fatty acids to triglycerides such asstearoyl-CoA-desaturase (SCDI) or the insulin responsive glucosetransporter (GLUT4). The product of the ob gene, leptin is a 16,000molecular weight polypeptide that is only expressed in pre-adipose cellsor adipose tissue. The expression of CCAAT enhancer binding protein,C/EBP, has been shown to precede the expression of several markers ofadipogenic differentiation and it is thought to play a key role inadipocyte development. Another marker is 422 adipose P2 (422/aP2), aprotein whose expression is enhanced during adipocyte differentiation(Cheneval, et al, 1991.). This differentiation pathway is thought alsoto involve peroxisome proliferation-activated receptor γ2 (PPAR γ2),which is involved in the initiation of transcription of adipocyte genes(Tontonoz, et al, 1994). Fatty acids are known activators of PPAR γ(Forman, et al., 1995). Studies using these markers and the describedmethods will allow a more detailed analysis of the lineage progressionof mesenchymal stem cell to adipocyte differentiation.

Lipid Soluble Dyes as Markers of Adipocyte Differentiation

Lipid soluble dyes are available to stain lipid vaculoes in adipocytes.These include Nile Red, Nile Blue, Sudan Black and Oil Red O, amongothers. Each of these hydrophobic dyes has a propensity to accumulate inthe lipid containing vaculoes of the developing adipocytes and canreadily identify the adipogenic cells in populations of differentiatingMSCS. At least one of these dyes can be used to isolate adipocytes fromnon-differentiated cells using a fluorescence activated cell sorter(FACS). An example of the use of Nile Red to identify adipogenic hMSCsis shown in FIG. 4.

EXAMPLE 1 Generation of Adipocytes from Human MSCs

Human MSCs are isolated from the red bone marrow of volunteer donors asdescribed in U.S. Pat. No.5,486,359. The cells are grown until coloniesare well established and at this point the cells are subcultured (1 to3) or they can be taken to assay for in vitro adipogenesis. For theadipogenesis assay, hMSCs are subcultured into 35 mm tissue culturedishes at 100,000 cells per dish and fed with 2 milliliters normal hMSCMedia (Dulbecco's Modified Eagle's Media (DMEM), 10% selected FetalBovine Serum (FBS) and antibiotic/antimycotic mixture (IX) (LifeTechnologies, Inc.)) and cells are maintained at 37° C., 5% CO₂ and 90%humidity. The cells are refed with the fresh media every third day andare allowed to multiply and become confluent. The cells are maintainedafter reaching confluence by refeeding every third day and this timeperiod of post confluence culturing enhances the adipogenic response inthe next step (at least out to 14 days). The differentiation intoadipocytes is initiated by changing the media to 2 ml of AdipogenicInduction Media (DMEM with 10% fetal bovine serum containing 10 μg/mlinsulin (human recombinant, Boehringer Mannheim Corp.), 0.5 mM methylisobutylxanthine (MIX)(Sigma Chemical Co.), 1 μM dexamethasone(Dex)(Sigma Chemical Co.)). This media is left on the cells for 48 hrswith cells maintained at 37° C., 5% CO₂, 90% humidity and is thenreplaced with Adipogenic Maintenance Media (DMEM containing 10% FBS and10 μg/ml insulin). The medium is changed every 3-4 days. The hMSCs beginto show small lipid vacuoles in 3-7 days and these enlarge and becomemore numerous over time, out to at least 30 days. There are severalvariations that have been successfully tried.

When human MSCs are treated as described above, they undergodifferentiation to the adipogenic lineage, as shown in FIG. 1. FIG. 1Ashows hMSCs (4×) cultured in normal hMSC media for the same period oftime as FIG. 1B. There is no evidence of lipid containing vacuoles andthe cells maintain the appearance of fibroblasts at high density. InFIG. 1B are hMSCs that were allowed to become confluent and thenmaintained for 10 days prior to adding the Adipogenic Induction Mediafor 48 hrs, and then changed to the Adipogenic Maintenance Media for anadditional 2 weeks. The lipid vacuoles are first apparent at about 3-7days but increase in size and abundance over time. FIG. 2A shows asimilar control culture as FIG. 1A at higher magnification (20×). FIG.2B show a culture of confluent hMSCs that were subjected to AdipogenicInduction Media for 48 hours and then maintained in AdipogenicMaintenance Media for 14 days. The many lipid containing vacuoles ofadipocytes are evident in a large proportion of the cells.

FIG. 3 shows the results of culturing hMSCs under a variety ofconditions, only one of which shows a high degree of adipogenicdifferentiation. All photos are at 10× magnification. FIG. 3A shows aculture of hMSCs maintained in normal hMSC culture media with noadditives. The cells grow with a fibroblastic morphology. FIG. 3B showsa similar culture that was treated with Adipogenic Induction Media for48 hours and then with Adipogenic Maintenance Media for an additional 14days with media changes every 3 days. The adipogenic cells, perhaps asmany as 30-35% of the cells, are evident as they contain the largerefractile lipid vacuoles. FIG. 3C shows a culture of hMSCs that weremaintained in the Adipogenic Maintenance Media for 14 days but was neversubjected to the dexamethasone/methyl isobutylxanthine treatment. Thecells maintain a flat morphological appearance with no evident vacuoles.FIG. 3D shows a culture of hMSCs that were treated with normal hMSCmedia containing 1 μM dexamethasone for 48 hours and then cultured for14 days in the Adipogenic Maintenance Media. The cells are disorganizedbut show very few, if any, lipid vacuoles. FIG. 3E shows a culture ofhMSCs that was treated with normal hMSC containing 0.5 ml. methylisobutylxanthine during the induction period and then was maintained for14 days in the Adipogenic Maintenance Media. The cells retain a flatfibroblastic phenotype. FIG. 3F shows a culture of hMSCs that wastreated with a media that induces the cells along a osteogenic pathway.This media contains 0.1 μM dexamethasone, 10 mM β-glycerol phosphate and50 μM ascorbic acid 2-phosphate. The presence of refractile osteoidmaterial is evident but no large lipid vacuoles.

The adipogenic cells can also be identified using vital dyes andhistological stains that label the lipid vacuoles. FIG. 4A shows aculture of hMSCs subjected to the adipogenic treatment and cultured for14 days in the Adipogenic Maintenance Media. The large lipid vacuolesare evident in this bright field image. But the lipids can also berevealed by using a fluorescent lipid soluble dye such as Nile Red(Greenspan, et al. 1985) and viewing by epifluorescence illumination asshown in FIG. 4B.

The results shown here have been reproduced several times with hMSCsderived from different donors and additional information on the methodis described here. Similar results have been obtained with hMSCs fromall individuals tested (4 or more donors). The percentage of cells thatbecome lipid containing adipocytes varies depending on the specifics ofculturing. Specifically, those cells that were allowed to becomecompletely confluent and maintained this way for up to 2 weeks prior toadipocyte induction, showed a much higher percentage of adipocytesovertime than cultures that were induced prior to confluence or atconfluence. As many as 30-35% of the hMSCs appear adipogenic at 2 weekspost induction when treated as described herein as shown in FIG. 1BhMSCs can be cultured in various sizes of culture ware with equalsuccess so obtaining larger numbers of cells should not present anyproblem.

EXAMPLE 2 Enhancing Adipogenic Differentiation

These experiments were performed to determine whether a population ofhuman mesenchymal stem cells growing in culture can be treated to induceadipogenic differentiation (which induces a percentage of 5-10% of thecells to become adipocytes), and then retreated at a later time toinduce more of the hMSCs to differentiate. Experiments were alsoperformed to examine whether it is possible to purify a population ofinduced adipogenic cells from the mixed culture of hMSCs and adipogenicMSCs that result from the treatment with adipogenic agents. Both sets ofexperiments were successful as described below and in the accompanyingfigures.

hMSCs were induced to the adipogenic phenotype by the culturing inAdipogenic Induction Media for 48 hours as described. The media was thenchanged to Adipogenic Maintenance Media and cells were cultured at 37°C. in a 5% CO₂ atmosphere for 3 to 6 days until there were noticeablelipid droplets visible within cells. Approximately 5-10% of the cellsbecame adipogenic as seen in FIG. 5. FIG. 5A shows hMSCs in culturewhich were not treated with either adipogenic medium but which werecultured, fixed and stained at the same time as the adipogenic culturesshown in 5B and 5C. FIG. 5B was treated once with Adipogenic InductionMedia and then cultured in Adipogenic Maintenance Media for anadditional three weeks and then fixed in neutral buffered formalin andstained with Oil Red O, a lipid soluble dye that accumulates in the fatdroplets. A dish of hMSCs was also retreated with fresh AdipogenicInduction Media for a second and third 48 hour period to induce morehMSCs to become adipogenic as shown in FIG. 5C. As many as 30-40% of thecells were converted to adipocytes by the three induction treatmentswhen viewed two weeks after the third treatment. Panels 5A-5C were allstained with Oil Red O.

EXAMPLE 3 Isolation of Adipocytes from hMSCs

The generation of adipocytes from human mesenchymal stem cells by theconditions described above produces large numbers of adipocytes, perhapsas many as 30%-40% of the cells present. For uses requiring a purepopulation, the adipocytes can be isolated from the non-adipogenic hMSCsby several methods as listed below.

Method one for isolating adipogenic hMSCs uses density gradientcentrifugation and takes advantage of the greater buoyancy of thelipid-containing adipogenic cells. In this method, cultures containinghMSCs and adipocytes derived from hMSCs are treated with 0.05%trypsin/0.53 mM EDTA to remove the cells from the culture dish and thecells are washed by adding 10 ml of normal hMSC media and centrifugedfor 10 minutes at 1000 rpm in the GS-6R centrifuge (Beckman Instruments,Inc.) at 20° C. The pelleted cells containing adipocytes and hMSCs areresuspended in 2 ml of the Adipogenic Maintenance Media and carefullylayered on top of 8 ml of PERCOLL of a density of 1.045 g/ml. The tubesare centrifuged at 2,200 rpm (1100 xg) in a Beckman GS-6R centrifuge for20 minutes at 3° C. The adipocytes are recovered in the uppermost 2 mlsand at the interface with the 1.045 density PERCOLL. (The non-adipogenicMSCs enter into the 1.045 density PERCOLL and can be recovered at thebottom of the tube.) The recovered adipocytes are washed by addition of10 mls of the Adipogenic Maintenance Media and centrifuged at 1000 rpmfor 10 min at 20° C. in the GS-6R centrifuge. The adipocytes arereplated at a density of 150,000 cells per 35 mm dish in AdipogenicMaintenance Media and returned to the incubator.

Method two for isolating adipogenic hMSCs uses fluorescence activatedcell sorting (FACS). The hMSCs differentiating into adipocytes in aculture can be isolated by using a lipid soluble fluorescent dye, suchas Nile Red (10-100 μg/ml) to stain lipid vacuole-containing adipocytes.The culture is then treated with trypsin/EDTA as above to release thecells from the culture vessel and subjecting the mixed population tofluorescence activated cell sorting (FACS). The parameters on themachine are adjusted to select and recover adipogenic cells in thepopulation and they can be used directly or replated and cultured in theincubator.

As described below, Method three of isolating the adipogenic cells in amixed population is to trypsin/EDTA treat and wash the cells as above.The cells are then placed in a tissue culture flask and the flask isfilled with media. The flask is closed tightly and turned upside-down sothat the surface treated for cell adhesion is uppermost. The buoyant,lipid-droplet-containing adipocytes rise to top and attach to thesurface of the flask. The next day the media is removed and the flaskrinsed with fresh media and the flask turned right-side-up. The flask,now with only enough media to cover the cell layer, is returned to theincubator for further maintenance.

Adipogenic hMSCs accumulate lipid droplets which decreases the bouyantdensity of the cells. The adipogenic hMSCs were then isolated asfollows. The dish of cells containing adipocytes and non-adipogenichMSCs from a culture that were treated for only one 48 hour inductionperiod and then grown for three weeks, was treated with 0.05% trypsinand 5 mM EDTA for 3-5 minutes to release the cells from the dish. Thecells were then rinsed from the dish with 10 ml of fresh media andplaced in a 25 cm² flask and the flask was filled to the brim with freshmedia The flask was turned upside down so the usual culture surface wasuppermost and the flask placed in the 37° C. incubator overnight. Themore bouyant adipogenic cells rose to the top and attached to thesurface, while the non-adipogenic hMSCs settled to the bottom surfaceand attached. The following day photos were taken of the cells on eachsurface as shown in FIG. 6. FIG. 6a shows the adipogenic hMSCs thatattached to the uppermost surface. The population is composed of greaterthan 99% adipogenic cells as evidenced by the lipid droplets in everycell. FIG. 6b shows the non-adipogenic hMSCs that settled to the lowersurface of the flask. Very few cells containing lipid droplets werepresent on the lower surface. These non-adipogenic cells could betreated with trypsin/EDTA and replated to another dish and be shown toretain adipogenic potential (data not shown), indicating that theyremain as mesenchymal stem cells, capable of lineage progression.

EXAMPLE 4

These experiments were performed to determine the effect of addingvarying concentrations of indomethacin to mesenchymal stem cells inculture to induce adipogenic differentiation. Test conditions includedindomethacin at 50, 100, 200 μM added to 10⁻⁷ M dexamethasone, 0.5 mMmethyl isobutylxanthine, 10 μg/ml insulin in DMEM (w/ 4.5 g/l glucose);three 48 hour treatments with 24-48 hours in between in DMEM (4.5 g/lglucose) with 10 μg/ml insulin: cultures were then allowed to accumulatelipid for another one week in DMEM (4.5 g/l glucose) with insulin beforefixation for histology and assessment of results. We also testedindomethacin under the same conditions however in the absence of methylisobutylxanthine.

The results were compared visually by observing the accumulation oflipid within the cells and by staining the lipid with the fluorescentlipid-soluble dye Nile Red and reading on a fluorescence plate reader asdescribed in Example 6.

The results provided in Table 1 show that the inclusion of indomethacinin the MDI induction medium resulted in a dose dependent increase in thecommitment of the hMSCs tested to the adipocyte lineage and an increasein the accumulation of intracellular lipid in the culture. Indomethacinat 200 μM in MDI gave the largest increase in adipocytes as shown bynearly 100% of the hMSCs becoming adipocytes. The Nile Red staining asdetermined by the fluorescent plate reader was nearly three times thatof the MDI sample alone. Indomethacin also increased the adipogenicdifferentiation of hMSCs in DMEM containing dexamethasone and insulinonly (DI), but to a level approximately half that of theMDI+indomethacin sample. Indomethacin with insulin had no significanteffect on hMSC adipogenesis.

TABLE 1 DAPI Nile red Nile red/ Sample Condition (cor) (cor) DAPI 1Control 8.84 0.65 0.07 2 Adipo maintenance 5.79 0.77 0.18 3 MDI (MIX inH2O) 7.5 6.97 0.93 4 MDI (MIX in DMSO) 7.31 7.25 0.99 5 MDI + 50 μMindomethacin 7.8 11.72 1.50 6 MDI + 100 μM indomethacin 7.11 16.39 2.317 MDI + 200 μM indomethacin 8.72 20.27 2.32 8 DI + 50 μM indomethacin6.66 3.11 0.47 9 DI + 100 μM indomethacin 6.77 5.35 0.79 10 DI + 200 μMindomethacin 6.03 10.08 1.67 11 I + 50 μM indomethacin 5.12 0.64 0.13 12I + 100 μM indomethacin 5.81 0.74 0.13 13 I + 200 μM indomethacin 5.280.74 0.14 cor = corrected (minus background) DAPI =4,6-Diamino-2-phenylindole

EXAMPLE 5

These experiments were performed to determine the effect of addingvarying concentrations of 15-deoxy Δ^(12,14) -prostaglandin J₂(15d-PGJ₂) to mesenchyrnal stem cell in culture to induce adipogenicdifferentiation. Test conditions included 1 or 10 μM 15d-PGJ₂ added to10⁻⁷ M dexamethasone, 0.5 mM methyl isobutyixanthine, 10 μg/ml insulinin DMEM (w/ 4.5 g/l glucose); three 48 hour treatments with 24-48 hoursin between in DMEM (4.5 g/l glucose) with 10 μg/mi insulin: cultureswere then allowed to accumulate lipid for another one week in DMEM (4.5g/l glucose;) with insulin before fixation for histology and assessmentof results. We also tested 15d-PGJ₂ under the same conditions withoutmethyl isobutylxanthine present.

The accumulation of lipid within the cells was observed visually and bystaining the lipid with the fluorescent lipid-soluble dye Nile Red andreading on a fluorescence plate reader as described in Example 6. Theresults provided in Table 2 show that for the MSCs tested, the inclusionof 15-deoxy-Δ^(12,14)-prostaglandin J₂ in the NMDI induction mediumresulted in a dose dependent increase in the adipogenic response and, at10 μM, it was nearly as effective as indomethacin when added to the MDImedium. It had no significant effect when included with insulin alone.Treatment with DI+15d-PGJ₂ gave a weak adipogenic response.

TABLE 2 DAPI Nile red Sample Condition (cor) (cor) Nile red/DAPI 1Control 4.71 0.79 0.17 2 Adipo maintenance 4.96 0.85 0.17 3 MDI 6.676.91 1.04 4 MDI + 1 μM 15d-PGJ2 6.53 13.16 2.02 5 MDI + 10 μM 15d-PGJ27.22 17.56 2.43 6 DI + 1 μM 15d-PGJ2 5.99 1.19 0.20 7 DI + 10 15d-PGJ25.36 2.57 0.48 8 I + 1 μM 15d-PGJ2 4.98 0.83 0.17 9 I + 10 μM 15d-PGJ25.75 0.85 0.15 cor = corrected (minus background) DAPI =4,6-Diamino-2-phenylindole

EXAMPLE 6 Assay

hMSCs were grown in 12 or 24 well tissue culture plates and were treatedwith adipogenic inducing agents as described above when the cells werepost-confluent. After three treatments, the cells were allowed toaccumulate lipid for an additional week prior to analysis. The celllayers were rinsed with Dulbecco's modified PBS (D.PBS) and fixed for 30minutes in 10% Neutral Buffered Formalin. The plates were then rinsedwith D. PBS and incubated for 30 minutes with 0.02% Saponin, 0.8 μg/mlDAPI (from an aqueous 2 mg/ml stock) and 1 μg/ml Nile Red (from a 1mg/ml stock in acetone) in D. PBS. Plates were then rinsed three timeswith D.PBS. The plates were read on a Molecular Devices FluorescencePlate Reader with the 355/460 nm filter set for DAPI and 485/538 nmfilter set for Nile Red and results displayed as relative fluorescenceintensity.

EXAMPLE 7 GELFOAM can Support In Vitro Differentiation of hMSCs IntoAdipocytes

GELFOAM sponge (size 100) (Upjohn, Kalamazoo, Mich.) was cut into smallpieces (approximately 3 mm×2 mm×2 mm. The samples were hydrated in twochanges of MSC culture medium to adjust the pH of the material to thatof the medium. The GELFOAM was then blotted onto sterile gauze to removeexcess medium and placed on a sterile culture dish. A volume of mediumcontaining MSCs sufficient to fill the voids in the GELFOAM was added tothe GELFOAM (0.3 ml suspension containing 300,000 MSCs). The materialwas gently compressed several times to eliminate bubbles and allow thecell suspension to be drawn into the interstices. The GELFOAM with cellswas placed in the incubator and the cells were allowed to attach for 30minutes. The dish was then filled with enough culture medium to coverthe GELFOAM and the sample was placed back in the incubator to allow thecells to grow and populate the material further. Medium was changed on aregular basis, twice a week for two weeks. The medium was then changedto MDI medium to induce differentiation of MSCs to adipocytes. The MDImedium was added for 48 hours and then changed to MSC medium for 24hours. This regimen was repeated twice more and then the GELFOAM withcells was cultured for an additional week to allow lipid to accumulatein the adipocytes. Nile red was added to the culture medium for 30minutes to stain the lipid vacuoles in the adipocytes. The sample wasobserved on an inverted microscope using ultraviolet light and thefluorescence from the dye stained vacuoles recorded on film. An exampleof the fluorescently stained adipogenic MSCs in GELFOAM is shown in FIG.12.

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What is claimed is:
 1. A composition for producing adipocytes,comprising isolated human mesenchymal stem cells in a biocompatiblematrix which supports the differentiation and maturation of humanmesenchymal stem cells into adipocytes.
 2. The composition of claim 1,wherein the matrix is comprised of collagen or gelatin.
 3. Thecomposition of claim 1, and further comprising a substance in an amounteffective to induce said stem cells to differentiate into adipocytes.